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Methods in
Molecular Biology 2313

Gunnar Houen Editor

Therapeutic
Antibodies
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
Therapeutic Antibodies

Methods and Protocols

Edited by

Gunnar Houen
Department of Neurology, Rigshospitalet, Glostrup, Denmark
Editor
Gunnar Houen
Department of Neurology
Rigshospitalet
Glostrup, Denmark

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1449-5 ISBN 978-1-0716-1450-1 (eBook)
https://doi.org/10.1007/978-1-0716-1450-1

© Springer Science+Business Media, LLC, part of Springer Nature 2022

This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and
retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter
developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to
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expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been
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Cover Illustration Caption: Space-filling model of Infliximab, the first therapeutic antibody to be approved for human
use, in ”open” conformation. Infliximab binds tumor necrosis factor (TNF) and is used for treatment of rheumatoid
arthritis and several other inflammatory diseases. From Maibom-Thomsen SL, Trier NH, Holm BE, Hansen KB,
Rasmussen MI, Chailyan A, Marcatili P, Højrup P, Houen G. Immunoglobulin G structure and rheumatoid factor
epitopes. PLoS One. 2019;14:e0217624.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Dedication

In memory of my late, beloved wife Eva.

v
Preface

Therapeutic antibodies (biologics) are the fastest growing pharmaceutical drug group and
have had tremendous clinical and scientific impact in cancer, autoimmune diseases, infec-
tious diseases, and other immune-related diseases. Therefore, methods for measuring the
concentration of therapeutic antibodies, determining development of (neutralizing) anti-
bodies to the drugs (which themselves are antibodies), and for predicting and monitoring
the therapeutic efficacy of individual drugs are of immense interest.
This volume of the Methods in Molecular Biology series covers methods for studying,
producing, and analyzing therapeutic antibodies, measuring their concentration, developing
neutralizing antibodies for them, and for predicting their clinical effects. It is intended to be
helpful for all persons working with the production of, research on, and development of
therapeutic antibodies as well as for clinicians using therapeutic antibodies in daily work with
patients.
I thank all contributors and editorial staff who have been helpful during the work. Also,
I thank all my students and collaborators through the years.
The volume is dedicated to the memory of my late, beloved wife Eva Thomsen
(29.11.1952–22.01.2021).
Finally, I am grateful to the series editor, John Walker, for support and for giving me the
opportunity to edit this volume of MiMB.

Glostrup, Denmark Gunnar Houen

vii
Contents

Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 Therapeutic Antibodies: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Gunnar Houen
2 Treatment of Autoimmune Diseases with Therapeutic Antibodies:
Lessons Learned from PID Patients Allow for Stratification
of the Infection Risk. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Joyce J. B. C. van Beers and Jan G. M. C. Damoiseaux
3 B-Cell Analysis for Monitoring Patients Undergoing B-Cell
Depletion for the Treatment of Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . 45
Jan G. M. C. Damoiseaux, Joyce J. B. C. van Beers,
Matthias Busch, and Pieter van Paassen
4 Assessment of Therapeutic Antibody Developability
by Combinations of In Vitro and In Silico Methods . . . . . . . . . . . . . . . . . . . . . . . . . 57
Adriana-Michelle Wolf Pérez, Nikolai Lorenzen,
Michele Vendruscolo, and Pietro Sormanni
5 The Therapeutic Antibody Profiler for Computational
Developability Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Matthew I. J. Raybould and Charlotte M. Deane
6 Short Read-Length Next Generation DNA Sequencing
of Antibody CDR Combinations from Phage Selection Outputs . . . . . . . . . . . . . . 127
Landon Pastushok, Kris Barreto, and C. Ronald Geyer
7 Large-Scale Transient Production in ExpiCHO-S™ with
Enhanced N-Galactosylation-Sialylation and PEI-Based Transfection . . . . . . . . . . 143
Xiaotian Zhong, Ashley Schwab, Weijun Ma, Caryl L. Meade,
Jing Zhou, Aaron M. D’Antona, Will Somers, and Laura Lin
8 Production of Therapeutic Single-Chain Variable Fragments
(ScFv) in Pichia pastoris. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Laia Montoliu-Gaya and Sandra Villegas
9 Purification of Therapeutic Antibodies by Protein A Affinity
Chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Anurag S. Rathore and Sharad Narnaware
10 Ion Exchange Chromatographic Methods for Purification
of Therapeutic Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Anurag S. Rathore and Vishwanath Hebbi
11 Structure-Indicated LC-MS/MS Bioanalysis of Therapeutic Antibodies . . . . . . . 187
Noriko Iwamoto and Takashi Shimada

ix
x Contents

12 Use of PASEF for Accelerated Protein Sequence Confirmation

and De Novo Sequencing with High Data Quality. . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Detlev Suckau, Waltraud Evers, Eckhard Belau, Stuart Pengelley,
Anja Resemann, Wilfred Tang, K. Ilker Sen, Elsa Wagner,
Olivier Colas, and Alain Beck
13 Nano-Microscopy of Therapeutic Antibody Aggregates in Solution . . . . . . . . . . . 219
Yukako Senga, Toshihiko Ogura, Hiroshi Imamura, and Shinya Honda
14 Measuring Self-Association of Antibody Lead Candidates
with Dynamic Light Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Fabian Dingfelder, Anette Henriksen, Per-Olof Wahlund,
Paolo Arosio, and Nikolai Lorenzen
15 Intact Mass Quantitation of Therapeutic Antibodies
for Pharmacokinetic Studies Using Immuno-Purification . . . . . . . . . . . . . . . . . . . . 259
Lisa A. Vasicek, Daniel S. Spellman, and Kevin P. Bateman
16 Detection of Residual Host Cell Proteins in Biotherapeutic
Drugs by Concatenated 2D LC-MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Feng Yang, Delia Li, and Donald E. Walker
17 Bioassays for the Evaluation of Target Neutralization
and Complement-Dependent Cytotoxicity (CDC)
of Therapeutic Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Nohemı́ Salinas-Jazmı́n, Emilio Medina-Rivero,
and Marco Antonio Velasco-Velázquez
18 Methods for Functional Characterization of FcRn Interactions
with Therapeutic Antibodies and Fc-Fusion Proteins . . . . . . . . . . . . . . . . . . . . . . . . 295
Shan Chung, Yuwen L. Lin, Van Nguyen, and Chang Liu
19 Method for Measurement of Antibody-Dependent Cellular Phagocytosis . . . . . . 305
Lynn Kamen, Benjamin Ordonia, Srividya Myneni,
and Shan Chung
20 Bioluminescent Bridging Immunoassay for Anti-Drug Antibody
(ADA) Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Nidhi Nath, Becky Godat, Rod Flemming, and Marjeta Urh
21 A Surface Plasmon Resonance-Based Assay for Simultaneous
Measurement of Serum Concentrations of Therapeutic
Antibodies and Anti-Drug Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Marten Beeg, Eleonora Allocati, and Marco Gobbi

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
Contributors

ELEONORA ALLOCATI • Center for Health Regulatory Policies, Istituto di Ricerche

Farmacologiche Mario Negri IRCCS, Milan, Italy
PAOLO AROSIO • Department of Chemistry and Applied Biosciences, Institute for Chemical
and Bioengineering, Swiss Federal Institute of Technology, Zurich, Switzerland
KRIS BARRETO • Department of Pathology and Lab Medicine, University of Saskatchewan,
Saskatoon, SK, Canada
KEVIN P. BATEMAN • Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck &
Co., Inc., West Point, PA, USA
ALAIN BECK • Biologics CMC and Developability, Institut de Recherche Pierre Fabre
(IRPF)—Centre d’Immunologie Pierre-Fabre (CIPF), Saint-Julien-en-Genevois, France
MARTEN BEEG • Laboratory of Pharmacodynamics and Pharmacokinetics, Istituto di
Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy
ECKHARD BELAU • Bruker Daltonics, Bremen, Germany
MATTHIAS BUSCH • Department of Internal Medicine, Maastricht University Medical
Center, Maastricht, The Netherlands
SHAN CHUNG • Department of BioAnalytical Sciences, Genentech Inc., South San Francisco,
CA, USA
OLIVIER COLAS • Biologics CMC and Developability, Institut de Recherche Pierre Fabre
(IRPF)—Centre d’Immunologie Pierre-Fabre (CIPF), Saint-Julien-en-Genevois, France
AARON M. D’ANTONA • BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D,
Cambridge, MA, USA
JAN G. M. C. DAMOISEAUX • Central Diagnostic Laboratory, Maastricht University Medical
Center, Maastricht, The Netherlands
CHARLOTTE M. DEANE • Oxford Protein Informatics Group, Department of Statistics,
University of Oxford, Oxford, UK
FABIAN DINGFELDER • Department of Biophysics and Injectable Formulation 2, Global
Research Technologies, Novo Nordisk A/S, Måløv, Denmark
WALTRAUD EVERS • Bruker Daltonics, Bremen, Germany
ROD FLEMMING • Promega Corporation, Madison, WI, USA
C. RONALD GEYER • Department of Pathology and Lab Medicine, University of
Saskatchewan, Saskatoon, SK, Canada
MARCO GOBBI • Laboratory of Pharmacodynamics and Pharmacokinetics, Istituto di
Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy
BECKY GODAT • Promega Corporation, Madison, WI, USA
VISHWANATH HEBBI • Department of Chemical Engineering, Indian Institute of Technology
Delhi, New Delhi, India
ANETTE HENRIKSEN • Department of Modelling and Predictive Technologies, Global Research
Technologies, Novo Nordisk A/S, Måløv, Denmark
SHINYA HONDA • Biomedical Research Institute, National Institute of Advanced Industrial
Science and Technology (AIST), Tsukuba, Ibaraki, Japan
GUNNAR HOUEN • Department of Neurology, Rigshospitalet, Glostrup, Denmark

xi
xii Contributors

HIROSHI IMAMURA • Biomedical Research Institute, National Institute of Advanced

Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan; Department of
Applied Chemistry, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan
NORIKO IWAMOTO • Shimadzu Bioscience Research Partnership, Shimadzu Scientific
Instruments, Bothell, WA, USA; Shimadzu Corporation, Kyoto, Japan
LYNN KAMEN • Department of BioAnalytical Sciences, Genentech Inc., South San Francisco,
CA, USA
DELIA LI • Protein Analytical Chemistry, Genentech, South San Francisco, CA, USA
LAURA LIN • BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge,
MA, USA
YUWEN L. LIN • Department of BioAnalytical Sciences, Genentech Inc., South San Francisco,
CA, USA
CHANG LIU • Department of BioAnalytical Sciences, Genentech Inc., South San Francisco,
CA, USA
NIKOLAI LORENZEN • Department of Biophysics and Injectable Formulation 2, Global
Research Technologies, Novo Nordisk A/S, Måløv, Denmark
WEIJUN MA • BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge,
MA, USA
CARYL L. MEADE • BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D,
Cambridge, MA, USA
EMILIO MEDINA-RIVERO • Unidad de Desarrollo e Investigacion en Bioprocesos (UDIBI),
Escuela Nacional de Ciencias Biologicas, Instituto Politécnico Nacional, Ciudad de Mé
xico, Mexico
LAIA MONTOLIU-GAYA • Department of Psychiatry and Neurochemistry, Institute of
Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg,
Mölndal, Sweden
SRIVIDYA MYNENI • Department of BioAnalytical Sciences, Genentech Inc., South San
Francisco, CA, USA
SHARAD NARNAWARE • Department of Chemical Engineering, Indian Institute of Technology
Delhi, New Delhi, India
NIDHI NATH • Promega Corporation, Madison, WI, USA
VAN NGUYEN • Department of BioAnalytical Sciences, Genentech Inc., South San Francisco,
CA, USA
TOSHIHIKO OGURA • Health and Medical Research Institute, National Institute of
Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
BENJAMIN ORDONIA • Department of BioAnalytical Sciences, Genentech Inc., South San
Francisco, CA, USA
LANDON PASTUSHOK • Department of Pathology and Lab Medicine, University of
Saskatchewan, Saskatoon, SK, Canada
STUART PENGELLEY • Bruker Daltonics, Bremen, Germany
ANURAG S. RATHORE • Department of Chemical Engineering, Indian Institute of Technology
Delhi, New Delhi, India
MATTHEW I. J. RAYBOULD • Oxford Protein Informatics Group, Department of Statistics,
University of Oxford, Oxford, UK
ANJA RESEMANN • Bruker Daltonics, Bremen, Germany
NOHEMÍ SALINAS-JAZMÍN • Departamento de Farmacologı́a, Facultad de Medicina,
Universidad Nacional Autonoma de México, Ciudad de México, Mexico
 Contributors xiii

ASHLEY SCHWAB • BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D,

Cambridge, MA, USA
K. ILKER SEN • Protein Metrics Inc., Cupertino, CA, USA
YUKAKO SENGA • Biomedical Research Institute, National Institute of Advanced Industrial
Science and Technology (AIST), Tsukuba, Ibaraki, Japan
TAKASHI SHIMADA • Shimadzu Bioscience Research Partnership, Shimadzu Scientific
Instruments, Bothell, WA, USA; Shimadzu Corporation, Kyoto, Japan
WILL SOMERS • BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D,
Cambridge, MA, USA
PIETRO SORMANNI • Centre for Misfolding Diseases, Department of Chemistry, University of
Cambridge, Cambridge, UK
DANIEL S. SPELLMAN • Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck
& Co., Inc., West Point, PA, USA
DETLEV SUCKAU • Bruker Daltonics, Bremen, Germany
WILFRED TANG • Protein Metrics Inc., Cupertino, CA, USA
MARJETA URH • Promega Corporation, Madison, WI, USA
JOYCE J. B. C. VAN BEERS • Central Diagnostic Laboratory, Maastricht University Medical
Center, Maastricht, The Netherlands
PIETER VAN PAASSEN • Department of Internal Medicine, Maastricht University Medical
Center, Maastricht, The Netherlands
LISA A. VASICEK • Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck &
Co., Inc., West Point, PA, USA
MARCO ANTONIO VELASCO-VELÁZQUEZ • Departamento de Farmacologı́a, Facultad de
Medicina, Universidad Nacional Autonoma de México, Ciudad de México, Mexico;
Unidad Periférica de Investigacion en Biomedicina Traslacional, Facultad de Medicina,
Universidad Nacional Autonoma de México, Ciudad de México, Mexico
MICHELE VENDRUSCOLO • Centre for Misfolding Diseases, Department of Chemistry,
University of Cambridge, Cambridge, UK
SANDRA VILLEGAS • Department of Psychiatry and Neurochemistry, Institute of Neuroscience
and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Mölndal,
Sweden
ELSA WAGNER • Biologics CMC and Developability, Institut de Recherche Pierre Fabre
(IRPF)—Centre d’Immunologie Pierre-Fabre (CIPF), Saint-Julien-en-Genevois, France
PER-OLOF WAHLUND • Department of Biophysics and Injectable Formulation 2, Global
Research Technologies, Novo Nordisk A/S, Måløv, Denmark
DONALD E. WALKER • Protein Analytical Chemistry, Genentech, South San Francisco, CA,
USA
ADRIANA-MICHELLE WOLF PÉREZ • Centre for Misfolding Diseases, Department of Chemistry,
University of Cambridge, Cambridge, UK
FENG YANG • Protein Analytical Chemistry, Genentech, South San Francisco, CA, USA
XIAOTIAN ZHONG • BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D,
Cambridge, MA, USA
JING ZHOU • BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge,
MA, USA
 Chapter 1

Therapeutic Antibodies: An Overview

Gunnar Houen

Abstract
Polyclonal immunoglobulin (Ig) preparations have been used for several decades for treatment of primary
and secondary immunodeficiencies and for treatment of some infections and intoxications. This has
demonstrated the importance of Igs, also called antibodies (Abs) for prevention and elimination of
infections. Moreover, elucidation of the structure and functions of Abs has suggested that they might be
useful for targeted treatment of several diseases, including cancers and autoimmune diseases. The develop-
ment of technologies for production of specific monoclonal Abs (MAbs) in large amounts has led to the
production of highly effective therapeutic antibodies (TAbs), a collective term for MAbs (MAbs) with
demonstrated clinical efficacy in one or more diseases. The number of approved TAbs is currently around
hundred, and an even larger number is under development, including several engineered and modified Ab
formats. The use of TAbs has provided new treatment options for many severe diseases, but prediction of
clinical effect is difficult, and many patients eventually lose effect, possibly due to development of Abs to the
TAbs or to other reasons. The therapeutic efficacy of TAbs can be ascribed to one or more effects, including
binding and neutralization of targets, direct cytotoxicity, Ab-dependent complement-dependent cytotox-
icity, Ab-dependent cellular cytotoxicity or others. The therapeutic options for TAbs have been expanded
by development of several new formats of TAbs, including bispecific Abs, single domain Abs, TAb-drug
conjugates, and the use of TAbs for targeted activation of immune cells. Most promisingly, current research
and development can be expected to increase the number of clinical conditions, which may benefit
from TAbs.

Key words Antibody, Antigen, Bispecific, Chimeric, Human, Humanized, Immunoglobulin, Mono-
clonal, Nomenclature, Therapeutic

1 Introduction

Antibodies (Abs) are natural anti-infectious agent molecules, which

react with foreign antigens (Ags) during infections (Eq. 1) and help
to neutralize pathogens and remove Ags [1–4]. Thus, Abs are
defined and named by their Ag specificity.
Ag þ Ab $ AgAb ð1Þ
Abs, also denoted immunoglobulins (Igs) are heterodimeric
proteins, which have two identical heavy chains (HCs) and two
identical light chains (LCs), linked by disulfide bonds (Fig. 1). Igs

Gunnar Houen (ed.), Therapeutic Antibodies: Methods and Protocols, Methods in Molecular Biology, vol. 2313,
https://doi.org/10.1007/978-1-0716-1450-1_1, © Springer Science+Business Media, LLC, part of Springer Nature 2022

1
2 Gunnar Houen

a b
Ag Ag
VH VH
VL VL

Fab: Specificity CH1 CH1 LC

CL CL

F(ab’)2

CH2 CH2

Fc: Effector funcons HC

CH3 CH3

Fig. 1 Basic architecture of human immunoglobulins (Igs). (a) An Ig is a symmetric heterodimeric molecule
with two identical parts linked by disulfide bonds. The molecule has two identical parts (“arms”) capable of
binding antigen (Ag). These are each denoted as a Fab (fragment antigen-binding). The last part of the Ig
molecule is denoted Fc (fragment constant or crystallizable) and contains the effector function sites of the
Ig. (b) The Ig molecule is composed of two identical light chains (LCs) and two identical heavy chains (HCs).
Each chain is a continuous polypeptide chain, folding into two (LC) or four (HC) domains. The LC domains are
denoted variable light (VL) and constant light (CL). The HC domains are denoted variable heavy (VH) and
constant heavy (CH) 1, 2, 3. The VL and VH domains together form the Ag-binding site and the part of this
determining the Ag specificity is called the complementarity-determining region (CDR). The part of the Ig
physically interacting with Ag is called the paratope and the part of the Ag binding directly to the Ig is called the
epitope

exist in several types, called IgM, IgD, IgA, IgG, and IgE, depend-
ing on the composition of their HCs, denoted μ, δ, α, γ, ε (Fig. 2)
[1–4]. The LCs can be of two types, kappa (κ) or lambda (λ) and
the HCs of IgA can be of two types (α1, α2) and of four types for
IgG (γ1–γ4) (see Notes 1 and 2). Thus, “Abs” is a collective name,
describing the antigen-binding property of Igs, which can be of
several types and isotypes described by the constituent LCs and
HCs (see Note 3). The three-dimensional structure of Abs has
traditionally been represented as an “open” Y, however, recent
studies have shown that Abs have a compact “closed” structure,
which opens upon contact with Ags [1, 5].
Antibodies are synthesized by B cells, which differentiate from
hemopoietic precursors and initially exist as “Ag-naı̈ve” cells with
membrane-associated IgM and IgD (with identical complementar-
ity-determining region (CDR) sequences and light chains).
Depending on the anatomic location and nature of the Ag, these
precursors switch to expression of IgG, IgA or IgE in response to
Ag stimulation and eventually differentiate to Ab-secreting plasma
cells (see Note 4), which take up residence in the bone marrow,
where their precursors originated [1, 6–10].
Vaccines induce Abs against foreign Ags, a phenomenon dis-
covered by early work on bacterial toxins and virus infections. For
example, immunization of animals with preparations of diphtheria
 Therapeutic Antibodies: An Overview 3

a c d
IgD, IgG, IgA

LC (κ/λ)

HC (δ/γ/α)
J
b
J
LC (κ/λ)

HC (μ/ε)
CH4 CH4

IgM, IgE IgA dimer IgM pentamer

Fig. 2 Structure of human immunoglobulin classes. (a) The basic structure of IgD, IgG and IgA is the same with
two light chains (LCs) and two heavy chains (HCs). The LCs are of κ or λ type in all three but the HCs are of δ, γ
or α type respectively. (b) IgA forms dimers of two identical IgA molecules joined by a separate chain, called
the joining chain (J). (c) The basic structure of IgM and IgE is the same with two LCs and two HCs. The LCs are
of κ or λ type in both, but the HCs are of μ or ε type, respectively, and has an additional constant domain
compared with the basic format shown in A. (d) IgM forms pentamers of the basic format joined by disulfide
bridges and a J chain

or tetanus toxins gave rise to formation of anti-toxins in the blood.

The anti-toxins were found to be associated with the Ig fractions of
plasma as revealed by electrophoretic separation of plasma compo-
nents, giving rise to the alternative name for Abs—Igs [1, 11, 12].

2 Immunoglobulin (Ig) Therapy

The importance of Abs for human health is illustrated by primary

(inherited) and secondary (acquired) Ig deficiencies, which may
lead to serious life-threatening conditions. Many of these condi-
tions can be treated or prevented by administration of purified Ig
preparations from healthy blood donor plasma, and such uses were
some of the first clinical applications of Ig therapy [1, 13–15].

2.1 Polyclonal Purified or semi-purified Ig preparations have been used for treat-
Human Ig Therapy ment and prevention of diseases for decades. Research on the
clinical use of normal human immunoglobulin (NHIG) was carried
out in the first and middle part of the twentieth century and NHIG
is now on the list of essential medicines issued by the World Health
Organisation (WHO) (see Note 5). Besides Ig deficiencies, NHIG
is used for treatment of several autoimmune diseases, and Ig
Another Random Scribd Document
with Unrelated Content
me to see Nazimova to-night! Oh!" She laughed aloud, and danced about
the spacious hall in her delight, while her sister, a very comely young
matron of thirty-five, leisurely removed her wraps.

"But Walter and I are going," Harriet casually remarked as she tossed her
cloak over a carved, high-backed chair. "The editor of the Bulletin gave
Walter two tickets as part payment for some legal business Walter did for
him. Of course you and I can't both be away from the children. Has the
baby had her five o'clock bottle?"

"It isn't quite five yet."

"Will you see that she gets it, dearie? I'm so dead tired, I'll have to rest
before dinner if I'm going into the city again to-night. Will you attend to it?"

"Yes."

"That's a dear. I'm going up to lie down. Don't let the children come to
my room and wake me, will you, dear?" she added as she started languidly
upstairs.

"But, Harriet!"

"What?" Harriet asked, not stopping.

"I accepted Aunt Virginia's invitation and she is coming out in her motor
for me!"

"Too bad! I'm awfully sorry. You'd better 'phone at once or she will be
offended. Tell her that as we are much too poor to buy tickets for the
theatre, we can't possibly refuse to use them on the rare occasions when
they're given to us!" Harriet laughed as she disappeared around the curve of
the winding stairway.

Margaret sprang after her. "Oh, Harriet! I can't give it up!" Her voice
was low and breathless.

"But if you 'phone at once Aunt Virginia won't be cross. You know,
dearie, you shouldn't make engagements without first finding out what ours
are." And Harriet moved on up the stairs to her bedroom.

Margaret was ashamed of her childishness when at dinner that evening

Walter, her brother-in-law, inquiring, in his kind, solicitous way, the cause
of her pallor and silence, she burst out crying and rushed from the table.

Walter, looking shocked and distressed, turned to his wife for an

explanation. But Harriet's face expressed blank astonishment.

"Why, I can't imagine! Unless she's tired out from having had the
children all day. I was at Mrs. Duncan's luncheon, you know. I didn't get
home until nearly five. I'll tell Margaret to go to bed early to-night and rest
up."

Walter Eastman, searching his wife's face keenly, shrugged his big
shoulders at the impenetrability of its innocent candour. No use to try to get
at the truth of anything from Harriet. She wasn't exactly a liar, but she had a
genius for twisting facts to suit her own selfish ends—and all Harriet's ends
were selfish. Even the welfare of her children was secondary to her own
comfort and convenience. Walter had no illusions about the wife of his
bosom and the mother of his three children. He knew perfectly well that she
loved no one as she loved herself, and that this dominating self-love made
her often cold-blooded and even sometimes a bit false, though always, he
was sure, unconsciously so. He was still quite fond of her, which spoke well
for them both, considering that they had been married nine years. Of course,
after such a length of time they were no longer "in love." But Harriet was an
easy-going, good-natured woman, when you didn't cross her; and as he was
also easy-going and good-natured, and never crossed her when he could
avoid it, they got on beautifully and had a pretty good time together.

Walter wondered sometimes what Harriet would do if placed in

circumstances where her own inclinations would have to be sacrificed for
those of another. For instance, if she and Margaret had to change places.

"Take Margaret to the play with you to-night and I'll stay home with the
kiddies, Harriet," he suggested, looking at his wife across their beautifully
appointed dinner-table with its old family china and silver. Harriet, in her
home-made evening gown, graced with distinction the stately dining-room
furnished in shining antique mahogany, its walls hung with interesting
portraits. "If Margaret's had charge of the children all day, she ought not to
have them to-night."

"No." Harriet shook her head. "Margaret ought not to go out to-night,
she's too tired. And I want you with me, dear. Margaret is not my husband,
you know. That's the danger of having one of your family living with you,"
she sighed. "It is so apt to make a husband and wife less near to each other.
I am always resisting the inclination, Walter, dear, to pair off with Margaret
instead of with you. I resist it for your sake, for the children's sake, for the
sake of our home."

"I shall feel a selfish beast going to a play and leaving that dear girl
alone here with the babies. They're our babies, not hers, you know."

"She loves them like her own; she's crazy about them. They are the
greatest pleasure she has, Walter."

"Because she hasn't the sort of young pleasures she ought to have. And
because she's so unselfish, Hat, that she lets herself be imposed upon to the
limit! I've been thinking, lately, that we ought to do more than we do for
Margaret; she ought to know girls of her own age; she ought to have a bit of
social life, now that the year of mourning is over. It's too dull for her,
sticking out here eternally, minding our children and seeing after the
house."

"But she's used to sticking out here and seeing after the house. When she
lived here with Uncle Osmond she had a lot less diversion and life about
her than she has now, and you know how deadly gloomy it was here then.
We've brightened it up and made it a home for Margaret."

"The fact that she had to sacrifice her girlhood for your uncle is all the
more reason why she shouldn't sacrifice what's left of it for our children."

"If Margaret doesn't complain, I don't see why you need, dear."

"She'd never complain—she never thinks of herself. Your Uncle Osmond

took care not to let her form the habit! For that very reason we should think
for her a bit, Hattie, dear. I say, we've got to let Margaret in for some young
society."

"When I can't afford to keep up my social end, let alone hers? And if we
should spend money that way for Margaret, where would the children come
in?"

"Oh, pshaw!" said Walter impatiently. "You're bluffing! You care no

more about the money side of it than I do. You're not a Yankee tight-wad!
Margaret need not live the life of a nursemaid because we're not rich, any
more than you do, honey. It's absurd! And it's all wrong. What you're really
afraid of, Hat, is that if she went about more, you'd have to stay at home
now and then with your own babies. Eh, dear?"

But he was warned by the look in his wife's face that he must go no
further. He was aware of the fact that Harriet was distinctly jealous of his
too manifest liking for Margaret. Being something of a philosopher, he had
felt occasionally, when his sister-in-law had seemed to him more than
usually charming and irresistible, that a wife's instinctive jealousy was
really a Providential safeguard to hold a man in check.

He wondered often why he found Margaret so tremendously appealing,

when undoubtedly his wife, though ten years older than her sister, was
much the better looking of the two. He was not subtle enough to divine that
it was the absolutely feminine quality of Margaret's personality, the
penetrating, all-pervasive womanliness which one felt in her presence,
which expressed itself in her every movement, in every curve of her young
body—it was this which so poignantly appealed to his strong virility that at
times he felt he could not bear her presence in the house.

He would turn from her and look upon his wife's much prettier face and
finer figure, only to have the fire of his blood turn lukewarm. For he
recognized, with fatal clearness, that though Harriet had the beautiful, clear-
cut features and look of high breeding characteristic of the Berkeley race,
her inexpressive countenance betrayed a commonplace mind and soul,
while Margaret, lacking the Berkeley beauty, did have the family look and
air of breeding, which gave her, with her countenance of intelligence and
sensitiveness, a marked distinction; and Walter Eastman was a man not only
of temperament, but of the poetic imagination that idealizes the woman
with whom he is at the time in love.

"The man that marries Margaret will never fall out of love with her—
she's magnetic to her finger-tips! What's more, there's something in her
worth loving—worth loving forever!"

At this stage of his reflections he usually pulled himself up short,

uncomfortably conscious of his disloyalty. Harriet, he knew, was wholly
loyal to him, proud of him, thinking him all that any woman could
reasonably expect a husband to be—a gentleman of old family, well set up
physically, and indeed good-looking, chivalrous to his wife, devoted to his
children, temperate in his habits, upright and honourable. She did not even
criticise his natural indolence, which, rather than lack of brains or
opportunity, kept his law practice and his earnings too small for the needs
of his growing family; but Harriet preferred to do without money rather
than have her husband be a vulgar "hustler," like a "Yankee upstart."

It was this same indolence of Walter's, rather than want of force of

character, which led him to stand by passively and see his sister-in-law
constantly imposed upon, as he distinctly felt that she was, though he
realized that Margaret herself, dear, sweet girl, never seemed conscious of
it. Her unexpected outburst at dinner to-night had shocked and hurt him to
the quick. He was sure that something really outrageous on Harriet's part
must have caused it. Yet rather than "raise a row" with Harriet, he
acquiesced in her decision to leave Margaret at home. It must be said in
justice to him that had his astute wife not kept him in ignorance of their
Aunt Virginia's invitation to Margaret he would undoubtedly have taken a
stand in the matter. Harriet, carefully calculating the limit of his easy
forbearance, knew better than to tell him of that invitation; and she could
safely count upon Margaret not to put her in the wrong with Walter.

Margaret, meantime, locked in her room, had quickly got over her
outbreak of weeping and was now sitting upright upon her bed, resolutely
facing her quandary.

It was Harriet's assumption of authority, with its implication of her own

subservient position, that was opening Margaret's eyes this evening to the
real nature of her position in her sister's household.

"Suppose I went straight to her just now, all dressed for the theatre, and
told her in an off-hand, careless, artistic manner that I couldn't possibly
break my engagement with Aunt Virginia!"

Margaret, perched Turk-fashion on the foot of her bed, her hands clasped
about one knee, her cheeks flushed, her eyes very bright, contemplated in
fancy Harriet's consternation at such an unwonted procedure on her part—
and she knew she would not do it. Not because, like Walter, she was too
indolent to wrestle with Harriet's cold-blooded tenacity; nor because she
was in the least afraid of her sister. After living eight years with Uncle
Osmond she would hardly quail before Harriet! But it was that thing Harriet
had said to her this afternoon—that awful thing that burned in her brain and
heart—it was that with which she must reckon before she could take any
definite stand. "You should not make any engagements without first finding
out what ours are," Harriet had said, which, in view of all the
circumstances, simply meant, "Being dependent upon us for your food and
clothes, your time should be at our disposal. You are no more free to go and
come than are the cook and butler."

Now of course Harriet would never admit for an instant that she felt like
that. Margaret knew perfectly well that her sister did not begrudge the little
it cost to keep her with them. Harriet was not so thrifty as that. This attitude,
then, was probably only a pretext to cover something else which Harriet
was no doubt unwilling to admit even to her own soul, that something else
which Margaret, herself, had tried so long not to see, which made her
presence at Berkeley Hill unwelcome to both Walter and Harriet. And
Harriet, too proud to acknowledge her true reason for wishing her sister
away, pretended to an economic one.

"Suppose I said to her, 'You must not make engagements without first
finding out what mine are?' Now if she had only said, 'We should not make
engagements without first consulting with each other.' But she put all the
obligation where she tries to persuade herself that it belongs."

When presently Margaret heard her sister and Walter leave the house to
go to the theatre she got up from her bed and went to Harriet's room
adjoining the nursery, to keep guard over the three sleeping children until
their parents came home.

Lying on a chintz-covered couch at the foot of Harriet's huge four-posted

bed, she thought long and earnestly upon every phase of her difficult
situation, determined that before she slept she would solve the apparently
impossible problem of how she might leave Berkeley Hill.

IV

Nine years ago it was that Margaret, a girl of sixteen, had come out from
Charleston to live at Berkeley Hill as nurse, amanuensis, housekeeper, and
companion to her sickly, irritable, and eccentric old Uncle Osmond
Berkeley, eminent psychologist, scholar, and author, who at that time owned
and occupied the Berkeley homestead. It was the death of her father and
Harriet's immediate marriage that, leaving her homeless and penniless, had
precipitated upon her those years of imprisonment with an irascible invalid.
Indeed so completely stranded had she been that she had accepted only too
thankfully her uncle's grudging offer to give her a home with him on
condition that she give him in return every hour of her time, making herself
useful in every variety of occupation he saw fit to impose, and to do it all
with entire cheerfulness and absolutely no complaining. That was the chief
of his many "unqualified conditions "—a cheerful countenance at all times,
no matter what her fancied reason for dissatisfaction, and no matter how
gloomy he might be.

"I'm never cheerful," he had affirmed, "and that's why I require you
always to be so. If that seems to you unreasonable and illogical, you're
stupid. Give the matter a little thought and light may come to you. You'll
have plenty of chance, living with me, to develop what little thinking
powers you may have—much more chance than you'd ever have in a school
for young ladies, where you no doubt think I ought to send you for the next
two or three years. Schools for young ladies! Ha!" he laughed sardonically.
"Ye gods! Thank me for rescuing you from the fate of being 'finished' at one
of them! Well named 'finishing schools!' They certainly are a girl's finish so
far as common sense, capacity for usefulness, and ability to think for herself
are concerned! And there actually are parents of daughters who seriously
regard such schools as institutions of 'education!' Yes, education, by God!
You'll get more education, my girl, from one week of my conversation than
you would from a decade of one of those parasite factories!"

It was in the library at Berkeley Hill, the stately old country home which
for seven generations had belonged to the Berkeley family, that this
preliminary interview had taken place, her uncle in his reclining chair
before a great open hearth, the firelight playing upon his pallid, intellectual
face crowned with thick, white hair, and upon the emaciated hands clasping
a volume on his knee. Repellently harsh he seemed to the shrinking maiden
standing before him in her deep mourning, to be inspected, appraised, and
catechised; for in spite of the fact that she had been born and brought up in
the city of Charleston, only two miles away, her uncle had never seen
enough of her to know anything about her.

Perceiving, now, how the girl shrank from him, his eyes sparkled; there
was something ghoulish in his love of cowing those who served him. For
the past ten years he had had no woman near him save hired attendants who
cringed before his bullying.

"A human creature who lets itself be bullied deserves no better," was his
theory, and he never spared a sycophant.

"The day I have you weeping on my hands," he warned his niece as she
stood pale and silent before him, "or even looking as though you were
trying not to weep, out you go!"

The fact that the girl was scarcely more than a child, that she was alone
and penniless, did not soften him.

"She's old enough to show her mettle if she has any. If she hasn't, no loss
if she's crushed in the grind of serving me, for I'm useful, and shall be while
I breathe and think."
 "Well, what have you to say for yourself, wench?" he demanded when
she had heard without a word his uncompromising statements as to what he
would require of her in return for the "home" he would give her.

"I accept all your unqualified conditions, Uncle Osmond," she answered
quietly, no tremor in her voice; and the musical, soft drawl of her tone fell
with an oddly soothing and pleasing effect upon the invalid's rasped nerves;
"if you'll accept my one condition."

Her uncle's white head jerked like a startled animal's. "What? What?" he
ejaculated after an instant's stunned silence. "Your condition? Huh! You
making a condition, upon my word! What pertness is this? A 'condition'
upon which you'll accept my charity!"

"Not your 'charity.' The self-supporting position of your cheerful,

uncomplaining, industrious, capable, untiring, companionable, intelligent
chattel," came the musical, lazy drawl in reply. "My condition is that you
solemnly promise never again to call me a 'wench.'"

"I'll call you what I see fit to call you! If you're so damned squeamish, I
won't have you near me! I'd be hurling books at your head!"

"I'm not 'damned squeamish,' Uncle Osmond, indeed I'm not. I really
rather like the way you swear, it's so manly and exciting. But I won't be
called a 'wench.'"

"Why not? I won't have my liberty of speech hampered!"

"Very well, then, Uncle Osmond, dear, I won't come."

"You shan't come! I wouldn't have you in the house, Miss Pernicketty!"

"Good-bye, then. I'm very sorry for you, Uncle Osmond. I'm sure the
loss is yours. I would have been very kind to you."

"Sorry for me! You think well of yourself, don't you, wench?"

"At least so well that I'll go out sewing by the day, or stand in a store, or
go on the stage, or turn evangelist (I've heard there's money in that) before
I'll be called a wench!"

"What in hell do you imagine the word means?"

"I don't know what it means, but I won't be addressed as a wench."

"Get the dictionary. Look it up."

"But I won't be called a wench no matter what it means."

"Won't be called one! You dictate to me? Understand, girl, nobody

dictates to me! Read Shakespeare's sonnet, Lucrece:

"'Know, gentle wench, it small avails my mood.'

No offence in the word, you see, my authority being our greatest English
poet."

"Good-bye, Uncle Osmond," she said, turning away and walking toward
the door.

"Come back and behave yourself!"

She came back at once. "All right—and don't ever forget your promise."

"I promised nothing. I never make promises."

"Your acceptance of my condition is a promise."

"Acceptance of your condition!" He choked and spluttered over it.

"And it's a mighty small condition considering all I'm going to do for
you with cheerfulness, amiability, a pleasant smile——"

"Hold your tongue and speak when you are spoken to!" he growled,
apparently furious, but secretly exulting at the child's refreshing
fearlessness with him.
 It had been an instinct of self-preservation that had led Margaret to
demonstrate to her uncle, in that very first hour with him, that the line
would have to be drawn somewhere in his browbeating. And the word
"wench" had served her purpose. Thereafter, in the eight years that she lived
with him, docile and patient as she always was, he never forgot, and she
never had to remind him, that there was a limit past which he could not
safely venture in the indulgence of his tendency to tyrannize.

But her life was hard; most girls would have found its monotony and
self-sacrifice unbearable; its gloomy environment in the great empty barn of
a house too depressing; its close confinement within the narrow limits of
the unkept grounds, overgrown with weeds and bushes, and dark with big
trees and a high hedge of hemlocks, as bad as any jail. There were
sometimes weeks at a stretch during which she saw no human being save
her uncle and the old negro couple who had lived on the place for a quarter
of a century; for though Harriet and her husband lived in Charleston, her
uncle would spare her so seldom to visit them, and was so exacting as to her
speedy return to him that she soon fell into the way of confining her
intercourse with her sister almost entirely to a weekly exchange of letters.

In spite, however, of her isolation Margaret felt that there were

compensations in her lot. She had resources within herself in her love of
books, and she found in her uncle's rich intellectual equipment, of which he
freely gave her the benefit in their daily association, a stimulus, a variety,
and even an excitement that meant much more to her than the usual girl's
diversions of frocks, parties, and beaus would have meant. It is true she
often longed for a congenial companion of her own age, she hungered for
affection, she suffered keenly in her occasional feverish paroxysms of
restlessness, and there were times when the surging fountains of her youth
threatened to break down the barriers that imprisoned a nature that was both
large and impassioned.

"She's temperamental enough!" was her uncle's early conclusion as, from
day to day, the girl's mind and heart were unfolded to his keen observation.

Her rare periods of passionate discontent, however, though leaving her

spent and listless for a time after they had passed over her, did not embitter
her. There was a fund of native sweetness in Margaret's soul that even her
life with cynical old Osmond Berkeley could not blight. That philosopher
marvelled often at his inability to spoil her, remarkably open as he found
her young mind to the ideas and theories which he delighted in impressing
upon her. It was indeed amazing how readily she would select from the
intellectual feast daily spread before her what was wholesome and pure and
reject what was morbid.

"That's right," he would approve when she would frankly refuse to

accept a dogma laid down to her. "Better think for yourself, even though
you think wrongly, than do as the other females of the species do—believe
whatever they are told to believe—or, worse, what it suits their personal
interests to believe. Be everlastingly thankful to me that I encourage you to
think for yourself, to face the facts of life. George Meredith writes, 'The
education of girls is to make them think that facts are their enemies.' You
shall not escape some knowledge of facts if I can help it!"

"It's awfully nice of you to care so much about my mind, Uncle

Osmond," she gratefully responded. "To really care for anything about me. I
do love to be mothered and coddled and made much of!"

"Huh! 'Mothered and coddled and made much of!' You're at the wrong
shop! And don't let me hear you misuse that word 'nice.'"

"I insist upon being pleased at your caring at least about my mind! I'd be
grateful even to a dog that was good to me."

"I'm not a dog, and I'm never so 'good' to any one that you could notice it
particularly."

"Don't try to make yourself out worse than you are; you're bad enough,
honey, in all conscience!"

"Hold your impudence and bring me Volume Third of Kant's 'Critique.'"

"Oh, dear!" Margaret sighed as she obeyed, "is it going to be that awful
dope to-day? I hoped up to the last you'd choose an exciting novel. Do you
know I don't think it's womanly to understand Kant's 'Critique.'"
 "I've no desire to be womanly. Do as I tell you."

In addition to finding his niece capable and patient as a nurse and

housekeeper, Margaret interested him more than any individual he had
known in many years. He secretly blessed the hour when she had come into
his sombre life to enliven and, yes, enrich it. Not for worlds, however,
would he have let her know what she was to him.

There were rare moments when he was actually moved to an expression

of gratitude and tenderness for his long-suffering victim; but Margaret's
touchingly eager response to such overtures (heart-hungry as she was in her
loneliness) while gratifying him, had always the effect of making him
promptly withdraw into his hard shell again and to counteract, by his most
trying exactions, his momentary softness; so that in time she learned to
dread any least sign of amiability.

She did not know the full extent of her uncle's selfishness in his
treatment of her: how ruthlessly he schemed to avert the danger which he
thought often threatened him of losing her to some one of the half-dozen
middle-aged or elderly gentlemen of learning who had the habit of visiting
him in his retirement and who, to the last man of them, whether married or
single, adored his niece. It seemed that no man could lay eyes on her
without promptly loving her (what men called love). Even his physician,
happily married and the father of four lusty boys, was, Berkeley could see,
quite mad about her, though Margaret never discovered it; she only thought
him extremely agreeable and kind and liked him accordingly. Indeed the
only fun she ever got out of this train of admirers was an occasional hour of
liberty while they were closeted with her uncle; for he took care, as soon as
he realized how alluring she was to most men, to have her out of the way
when his acquaintances dropped in, a deprivation to his own comfort for
which the visitor paid in an extra dose of pessimism and irony.

"When that child falls in love," Berkeley once told himself, "as of course
so temperamental a girl is bound to do sooner or later, it will go hard with
her. Let her wait, however, until I'm gone. Time enough for her then. I need
her. Couldn't endure life without her now that I'm used to her!"
 So he not only gave her no opportunity to meet marriageable men, he
tried to unsex her, to engraft upon her mind his own cynicism as to the thing
named love, his conviction of its gross selfishness, his scorn of
sentimentality and of "the hypocrisy that would idealize an ephemeral
emotion grounded in base, egoistic appetite."

"All 'love,' all attraction of whatever nature, is grounded in sex," he

would affirm. "The universe is upheld and constantly recreated by the
ceaseless action of so-called love. A purely natural, physical phenomenon,
therefore. There is not in life such a thing as a disinterested love."

"A mother's love?" Margaret once suggested in reply to this avowal.

"Entirely selfish. She loves her child as part of herself; all her pride and
ambition for it are because it is hers."

"Well, if you call a mother's love selfish, there's no use saying anything
more."

"And not to mince matters," he reaffirmed, "I want you to know for your
own protection that a man's love for a woman is that of a beast of prey for
its victim!"

"But I'm so safe here, I don't need such protection; I never see a man. No
one but learned scholars ever come here."

"'Learned scholars' are not men, then, in your category?"

"Not the interesting wild kind that you warn me against."

"The man, woman, or 'learned scholar,' who has not a devil as well as an
angel in his soul, a beast as well as a god, is too limited a creature to see life
whole and big and round."

"Am I, then," she inquired with interest, "a devil and a beast as well as
an angel and a goddess, do you think?"

"Mostly devil, you! I couldn't stand the angel-goddess combination.

Even you, my girl, are wholly selfish; you would not stay with me for one
day if it were not that I give you a home. Come, now," he invited, and
evidently expected a protest against this assertion.

"Why, of course I shouldn't. Why would I?"

He looked rather blank at this, though privately he never failed to find

her honesty refreshing.

"I never understood," she added, "that it was a question of affection

between you and me, did you, my dear?"

"'Affection!'" he sneered bitterly. "Affection for ourselves!"

"Of course. You wouldn't give me a bright and happy home like this if
you did not need me to wait on you thirty-six hours out of the twenty-four
with a cheerful, Cheshire-cat smile, and all for my food, bed, and two new
frocks and hats a year."

"Have you no appreciation, girl, of the liberal education it is for you to

be with me, to be permitted to read to me, to have such a library as mine at
your command?"

"Yes, indeed, Uncle Osmond."

"Well, then?"

"But I don't stay here for the pleasure of your amiable society, dear," she
assured him, patting his hand. "You're far too much like your old Scotch
Thomas Carlyle that you admire so much. My goodness, what a life Jane
must have led with that old curmudgeon!"

"Hold your impudent tongue!"

"Yes, dear."

"Don't speak to me again to-day!"

"Thanks; I'm so glad you don't also require me to be brilliantly

conversational. I'd really have to charge extra for that, Uncle Osmond."
 "Get me my eggnog!"

In spite of all Osmond Berkeley's precautions, however, Margaret did, of

course, go through the intense and fiery ordeal of "falling in love"; for when
a maiden's budding soul begins to unfold to the beauty of life, to throb and
thrill before the wonder and mystery of the universe, no walled
imprisonment can check the course of nature—she is bound to suffer the
bitter-sweet experience of becoming enamoured of something, it doesn't
much matter what; a cigar-shop Indian will suffice if nothing more lively
comes her way. For circumstances are, after all, nothing but "machinery,
just meant to give thy life its bent." Berkeley, priding himself on his
knowledge of sex-psychology, knowing that girls isolated in boarding-
schools fall in love with their woman teachers, and in colleges with each
other, nevertheless persuaded himself that he could, in this instance, defeat
nature; that Margaret was being safeguarded too absolutely to admit of her
finding any outlet whatever for the pent-up emotional current of her
womanhood.

But there came to Berkeley Hill one day a stranger, an earnest young
minister of Charleston, who, having read a magazine article of Osmond
Berkeley's in which "the hysterical, unwholesome excitement of
evangelistic revivals" was demonstrated to be purely physiological, wished
to remonstrate with its author and point out to him that he was grievously
mistaken.

One keenly appraising, glance at the embarrassed, awkward young man

as he was shown into the library where Berkeley sat in his armchair before
the fire, with Margaret at his side reading to him from a just published work
by Josiah Royce, made her uncle decide that it would be superfluous to
send her from the room—"on account of a creature like this, with no
manners, no brains, and an Adam's apple!"

But it was the young man's deadly earnestness in the discussion between
these two unequal protagonists that impressed itself upon Margaret's hungry
imagination; his courage in coming with what he conceived to be his
burning message of truth to such a formidable "enemy to truth" as the
famous scholar, Dr. Osmond Berkeley. Evidently, the young man's
conscience, in spite of his painful shyness, had lashed him to this visit,
more dreadful than a den of lions. There were still, even in these days, it
seemed, martyrs for religion.

Now, while Margaret of course recognized the intellectual feebleness of

the young minister's side of the question which was under fire, nevertheless,
before his visit was concluded, his brow wore for her a halo; his thin little
voice was rich music to her quivering nerves; his unsophisticated manner
the outward sign of a beautiful simplicity; his Adam's apple a peculiar
distinction.

Berkeley, as soon as he found his visitor a bore, made short work of him
and got rid of him without ceremony. In Margaret's eyes the young man
stood up to his rebuffs like a hero and a martyr.

Her uncle did not notice, upon her return to the library after seeing the
young man into the hall, how bright were her eyes, how flushed her cheeks,
how sensitive the curve of her lips.

"Ha, ha!" he laughed sardonically, "wouldn't you rather go to hell than

have to hear him preach?"

"You laugh like a villain in a melodrama!" retorted Margaret.

"I haven't laughed for twenty years except at damned fools. When did
you ever see a melodrama?"

"Aunt Virginia took Harriet and me to see The Two Orphans once."

"Damned presumption of the fellow to come here and take up my time!

He isn't even a gentleman."

"I thought you prided yourself on not being a snob, Uncle Osmond."

"Don't be stupid. Breeding is breeding."

"Well, what is good breeding if it isn't being courteous in your own

house? You may call that young man common, but I doubt whether he
bullies women!"
 "You're cross!" he snapped at her. "Look pleasant!" he commanded,
bringing his hand down heavily on the arm of his chair.

"I won't!" And for the first and only time in all the eight years of her life
with him, Margaret turned upon him with a stamp of her foot.

He stared at her incredulously.

"You call that good breeding, do you, stamping your foot at your
benefactor?"

"'Benefactor?'" Margaret flew across the room and violently turned the
pages of the dictionary on a stand in the corner. "'Benefactor,'" she read, '"a
doer of kindly deeds; a friendly helper.' You see, I'm your benefactor,
according to the Standard."

"You're begging the question: is it well-bred for a young lady to stamp

her foot?"

"I'm ashamed that I did it, Uncle Osmond, and I beg your pardon."

"Your tone is not contrite!" he objected. But an unwonted flash in her

eyes made him see that this was one of the places where he would have to
"draw the line."

"You are tired," he said abruptly. "No wonder, after listening to the
braying of that evangelical ass for nearly an hour! Put on your wraps and
take a run about the grounds."

As with a look of relief Margaret turned to leave the room, he added in a

tone that was almost gentle, "Put on your heavy coat, child, the air is very
raw."

"Thank you, Uncle Osmond."

"And come back looking cheerful."

"I shall have to turn Christian Scientist if I'm to be cheerful under all
circumstances—and you say you hate Christian Scientists because they are
always so damned pleasant."

"You can't turn Christian Scientist and live in the same house with me!"

"But, Uncle Osmond, dear, I'm beginning to see that a Christian Scientist
is the only thing that could live in the same house with you!"

With that she left him, to a half-hour of anxious consideration of her

final thrust; for the one dread that hung over his life was the possibility of
Margaret's deserting him.

Margaret's suddenly conceived passion for the young minister went

through all the usual phases. It was not, of course, the individual himself,
but her impossible inhuman ideal of him, of which she was enamoured, the
man himself was as unknown to her as though she had never seen him; his
image merely served as a dummy to be clothed with her rich imaginings.
The thought of him dwelt with her every moment of the day, making her
absent-minded and listless, or feverishly talkative. She made excuses to go
frequently to town, to a dentist, to a doctor, to see Harriet, just for a chance
to drive past the minister's parsonage, for even if she did not catch a
glimpse of him, it was manna to her soul to look upon the place of his
abode. She would have delighted to have lain her cheek upon the doorsill
his foot had pressed. The actual sight, once or twice, of his ungainly figure
on the street, set her heart to thumping so that she could not breathe. Her
discovery, through a paragraph in the religious news of a daily paper, that
he was married, did not affect her, for she was not conscious of any desire
to marry him; she only wanted to see him, to hear him, to feel herself alive
in all her being, in his presence.

Even the sermon she managed to hear him preach one Sunday morning,
when a visit from one of the scholarly gentlemen whom her uncle
considered dangerous, gave her a free half day, even her recognition,
through that sermon, of the man's mental barrenness, did not quench her
passion.

What did finally kill it, after three months of mingled misery and
ecstasy, was an occasion as trivial as that which had given birth to it. One
day, in front of a grocery shop, where some provisions were being piled into
her phaeton, and where, to her quivering delight, the Object of her adoration
just chanced at that moment to come to make some purchases, she heard
him say to a negro employee of the grocer, "Yes, sir, two pecks of potatoes
and a head of cabbage; no, sir, no strawberries."

To say "sir" to a negro! The scales fell from Margaret's eyes. Her heart
settled down comfortably in her bosom. Her nerves became quiet. The
young minister stood before her as he was. His Adam's apple was no longer
a peculiar distinction, but an Adam's apple. For this was South Carolina.

Thereafter, her uncle found her a much more comfortable companion.

But keenly observant though he was, he had never suspected for a moment,
during those three months of Margaret's obsession, that she was actually
experiencing the thing he was so persistently trying to avert; for it would
not have been conceivable to him that any woman, least of all his niece,
Margaret Berkeley, could fall in love with "a milksop" like "Rev. Hoops,"
as the poor man's printed visiting card proclaimed him.

Never in all the rest of her life could Margaret laugh at that youthful
ordeal. That she could have been so insanely deluded was a mystery to
wonder over, to speculate about; but the passion itself, the depth, the height,
the glory of it, its revelation of human nature's capacity for ecstasy—all this
was a reality that would always be sacred to her.

At the same time, her discovery that an emotional experience so intense

and vital, so fundamental, could grow out of an absolute illusion and be so
ephemeral, made her almost as cynical about love as was her uncle himself;
so that always after that the seed of skepticism, which he so earnestly
endeavoured to plant in her mind, fell on prepared soil.
 Had Margaret adopted indiscriminately her uncle's philosophical, ethical,
social, political, or even literary ideas, it would certainly have unfitted her
for living in a society so complacent, optimistic, and conventional as that of
most American communities. As it was, the opinions she did come to hold,
from her intercourse with this fearless, if pessimistic, thinker, and from her
wide and varied reading with him, and also the ideals of life she formed in
the solitude which gave her so much time for thought, were unusual enough
to make her unique among women. One aspect of this difference from her
kind was that she was entirely free from the false sentimentality of the
average young woman, and this in spite of the fact that she was fervently
imaginative and, in a high degree, sensitive to the beauty and poetry of life.
Another and more radical point of difference was that she had what so very
few women do have—spiritual and intellectual fearlessness. And both of
these mental attitudes she owed not only to her own natural largeness of
heart and mind, but to the strong bias given her by her uncle toward
absolute honesty.

While, by reason of her more than ordinary mentality, as well as because

of a very adaptable disposition, Margaret bore her life of self-sacrifice and
isolation with less unhappiness than most girls could have done, there was
one phase of it which was vastly harder upon her. Her nature being
unusually strong in its affections, it took hard schooling indeed before she
could endure with stoicism the loveless life she led. It was upon her relation
with her elder sister Harriet, the only human being who really belonged to
her, that she tried to feed her starved heart, cherishing almost with passion
this one living bond; idealizing her sister and her sister's love for her,
looking with an intensity of longing to the time when she would be free to
be with Harriet, to lavish upon her all her unspent love, to live in the
happiness of Harriet's love for her.

Harriet's lukewarmness, not manifest under her easy, good-natured

bearing, was destined one day to come as a great shock to Margaret.

It was one night about five months before her uncle's sudden death that
he talked with her of his will. They were together in the library, waiting for
Henry, the negro manservant, to finish his night's chores about the place
before coming to help the master of the house to bed.
 "I trust, Margaret," Berkeley, with characteristic abruptness, broke a
silence that had fallen between them, "that you are not counting on
flourishing as an heiress when I have passed out?"

"I must admit," said Margaret apologetically, "that I never thought of

that, stupid as it may seem to you, Uncle Osmond. Now that you mention it,
it would be pleasant."

"'Pleasant?' To have me die and leave you rich?"

"I mean only the heiress part would be pleasant—and having English
dukes marrying me, you know, and all that."

"How many English dukes, pray? I fancy they are a high-priced

commodity, and my fortune isn't colossal."

"I shouldn't want a really colossal fortune."

"Modest of you. But," he added, "if I did mean to do you the injury of
leaving you all I have, it would be more than enough to spoil what is quite
too rare and precious for spoiling"—he paused, his keen eyes piercing her
as he deliberately added—"a very perfect woman."

"Meaning me?" Margaret asked with wide-eyed astonishment.

"So I don't intend to leave you a dollar."

"Suit yourself, honey."

"You are like all the Berkeleys, entirely lacking in money sense. Now the
lack of money sense is refreshing and charming, but disastrous. I shall not
leave my money to you for four reasons." He counted them off on his long,
emaciated fingers. "First, because you wouldn't be sufficiently interested in
the damned money to take care of it; secondly, you'd give it away to your
sister, or to her husband, or to your own husband, or to any one that knew
how to work you; thirdly, riches are death to contentment and to usefulness
and the creator of parasitism; fourthly, I wish you to be married for your
good, sweet self, my dear child, and not for my money."
 "But if I'm penniless, I may have to marry for money. From what you tell
me of love, money is the only thing left to marry for. And if it has to be a
marriage for money, I prefer to be the one who has the money, if you please,
Uncle Osmond."

"Well, you won't get mine. I tell you you are worth too much to be
turned into one of these parasitical women who are the blot on our modern
civilization. In no other age of the world has there been such a race of
feminine parasites as at the present. Let me tell you something, Margaret:
there is just one source of pure and unadulterated happiness in life, and that
I bequeath to you in withholding from you my fortune. Congenial work, my
girl, is the only sure and permanent joy. Love? Madness and anguish.
Family affection? Endless anxiety, heartache, care. You are talented, child;
discover what sort of work you love best to do, fit yourself to do it
preëminently well, and you'll be happy and contented."

"But my gracious! Uncle Osmond, what chance have I to fit myself for
an occupation, out here at Berkeley Hill, taking care of you? These years of
my youth in which I might be preparing for a career I'm devoting to you,
my dear. So I really think it would only be poetic justice for you to leave me
your money, don't you?"

Her uncle, looking as though her words had startled and surprised him,
did not answer her at once. Considering her earnestly as she sat before him,
the firelight shining upon her dark hair and clear olive skin, the peculiar
expression of his gaze puzzled Margaret.

"That," he said slowly, "is an aspect of your case I had not considered."

"Of course you had not; it wouldn't be at all like you to have considered
it, my dear."

"Well," he snapped, "my will is made. I'm leaving all I have, except this
place, for the founding of a college which shall be after my idea of a
college. Berkeley Hill, however, must, of course, remain in the family."

"Don't, for pity's sake, burden the family (that's Harriet and me) with
Berkeley Hill, Uncle Osmond, if you don't give us the wherewithal to keep
it up and pay the taxes on it!" protested Margaret.

Again her uncle gazed at her with an enigmatical stare. "Huh!" he

muttered, "you've got some money sense after all. More than any Berkeley I
ever met."

"I know this much about money," she said sententiously: "that while
poverty can certainly rob us of all that is worth while in life, wealth can't
buy the two essentials to happiness—love and good health."

"Since when have you taken to making epigrams?"

"Why, that is an epigram, isn't it! Good enough for a copybook."

"I tell you, girl, if I leave you rich, I rob you of the necessity to work,
and that is robbing you of life's only worth. The most pitiable wretches on
the face of the earth are idle rich women."

"If it's all the same to you, Uncle Osmond, I'd rather take my chances for
happiness with riches than without them."

"I am to understand, then, that you actually have the boldness to tell me
to my face that you expect me to leave to you all I die possessed of?"

"Yes, please."

"It's wonderfully like your damned complacency! Well, as I've told you,
I've already made my will."

"Here's Henry to take you upstairs. But you can make it over, or add a
codicil. Which shall I bring you to-night, an eggnog or beer?"

"I'm sick of all your slops. Let me alone."

"Yes, dear. Good-night," she answered with the perfunctory, artificial

pleasantness which she always employed, as per contract, in responding to
his surliness; and the absurdity, as well as the audacity, of that bought-and-
paid-for cheerfulness of tone, never failed to entertain the old misanthrope.
 Five months later the will which Osmond Berkeley's lawyer read to the
"mourners" gave Berkeley Hill to Margaret and her sister, Mrs. Walter
Eastman, while all the rest of the considerable estate was left to a board of
five trustees to be used for the founding of a college in which there should
be absolute freedom of thought in every department, such a college as did
not then exist on the face of the earth.

Harriet's husband, being a lawyer, offered at once to secure for Margaret,

through process of law, a reasonable compensation for her eight years of
service. But Margaret objected.

"You see Uncle Osmond didn't wish me to have any of his money,
Walter."

"Don't be sentimental about it, Margaret. Your uncle had a lot of

sentiment, didn't he, about your sacrificing your life for him?"

"He had his reasons for not giving me his money. He sincerely thought it
would be better for me not to have it. He really did have some heart for me,
Walter. I'm not sentimental, but I couldn't touch a dollar he didn't wish me
to have."

"Then you certainly are sentimental," Walter insisted.

Almost immediately after the funeral Harriet and her family moved out
from Charleston to live at Berkeley Hill with Margaret, retaining the two
old negroes who for so many years had done all the work that was done on
the estate.

"We couldn't rent the place without spending thousands in repairing it, so
we'll have to live on it ourselves."

The sentiment that Margaret and Harriet cherished for this old
homestead which had for so long been occupied by some branch of the
family was so strong as to preclude any idea of selling the place.

It was Margaret's wish, at this time, to go away from Berkeley Hill and
earn her own living, as much for the adventure of it as because she thought
she ought not to be a burden to Walter. But the Southerner's principle that a
woman may with decency work for her living only when bereft of all near
male kin to earn it for her led Walter to protest earnestly against her leaving
their joint home.

Harriet, too, was at first opposed to it.

"You could be such a help and comfort to me, Margaret, dear, if you'd
stay. Henry and Chloe are too old and have too much work to do on this
huge place to help me with the children; and out here I can't do as I did in
Charleston—get in some one to stay with the babies whenever I want to go
anywhere. So you see how tied down I'd be. But with you here, I should
always feel so comfortable about the children whenever I had to be away
from them."

"But for what it would cost Walter to support me, Harriet, dear, you
could keep a nurse for the children."

"And spend half my time at the Employment Agency. A servant would

leave as soon as she discovered how lonesome it is out here, a half mile
from the trolley line. It's well Henry and Chloe are too attached to the place
to leave it."

"So the advantage of having me rather than a child's nurse is that I'd be a
fixture?" Margaret asked, hiding with a smile her inclination to weep at this
only reason Harriet had to urge for her remaining with her.

"Of course you'll be a fixture," Harriet answered affectionately. "Walter

and I are only too glad to give you a home."

So, for nearly a year after her uncle's death, Margaret continued to live at
Berkeley Hill.

Harriet always referred to their home as "My house," "My place," and
never dreamed of consulting her younger sister as to any changes she saw
fit to make in the rooms or about the grounds.
 It was during these first weeks of Margaret's life with Harriet that she
suffered the keen grief of finding her own warm affection for her sister
thrown back upon itself in Harriet's want of enthusiasm over their being
together; her always cool response to Margaret's almost passionate
devotion; her abstinence from any least approach to sisterly intimacy and
confidence. It was not that Harriet disliked Margaret or meant to be cold to
her. It was only that she was constitutionally selfish and indifferent.

So, in the course of time, Margaret came to lavish all the thwarted
tenderness of her heart upon her sister's three very engaging children.

But before that first year of her new life had passed over her head she
came to feel certain conditions of it to be so unbearable that, in spite of
Walter's protests (only Walter's this time), she made a determined effort to
get some self-supporting employment. And it was then that she became
aware of a certain fact of modern life of which her isolation had left her in
ignorance: she discovered that in these days of highly specialized work
there was no employment of any sort to be obtained by the untrained.
School teachers, librarians, newspaper women, even shopgirls,
seamstresses, cooks, and housemaids must have their special equipment.
And Margaret had no money with which to procure this equipment. There
is, perhaps, no more tragic figure in our strenuous modern life than the
penniless woman of gentle breeding, unqualified for self-support.

The worst phase of Margaret's predicament was that it had become

absolutely impossible for her to continue to live longer under the same roof
with Walter and Harriet. The simple truth was, Harriet was jealous of
Walter's quite brotherly affection for her—for so Margaret interpreted his
kindly attitude toward her. Having no least realization of her own unusual
maidenly charm, the fact that her brother-in-law was actually fighting a
grande passion for her would have seemed to her grotesque, incredible; for
Walter, being a Southern gentleman, controlled his feelings sufficiently to
treat her always with scrupulous consideration and courtesy. Therefore, she
considered Harriet's jealousy wholly unreasonable. Why, her sister seemed
actually afraid to trust the two of them alone in the house together!
(Margaret did not dream that Walter was afraid to trust himself alone in the
house with her.) And if by chance Harriet ever found them in a tête-à-tête,